Unlike adult differentiated tissue cells, pluripotent stem cells, such embryonic stem (ES) cells, can divide and self-renew indefinitely in-vitro and can also give rise to specialized cell types that can potentially form all tissues of the body (Evans, M. J. and Kaufman, M. H., Nature, 292(5819):154-156 (1981); Martin, G. R., Proc. Natl. Acad. Sci. USA, 78(12):7634-7648 (1981)). Therefore, the development of successful stem cell differentiation strategies to specific functional cell lineages offers the possibility of utilizing renewable cell sources to treat a large number of devastating conditions such as Parkinson's and Alzheimer's diseases, spinal cord injury, heart disease, diabetes, etc. (Cao, Q., et al., J. Neurosci. Res., 68(5):501-510 (2002); Dinsmore, J., et al., Cell Transplant., 5(2):131-143 (1996)). In addition to their applications in cell replacement, generation of mature cell types from stem cells could also provide materials for pharmacological and toxicological testing to improve the safety and efficacy of new drugs (O'Neill, A. and Schaffer, D. V., Biotechnol. Appl. Biochem., 40(Pt 1):5-16 (2004)). However, the enormous potential of stem cells relies on the effective generation of large numbers of functionally stable and homogenous differentiated cell populations. Although many investigators have described techniques to successfully differentiate stem cells into different mature cell lineages using growth factors or extracellular matrix protein supplementation (Bain, G., et al., Dev. Biol., 168(2):342-357 (1995); Kitazawa, A. and Shimizu, N., J. Biosci. Bioeng., 100(1):94-99 (2005); Okabe, S., et al., Mech. Dev., 59(1):89-102 (1996); Tian, H. B., et al., Acta Biochim. Biophys. Sin. (Shanghai), 37(7):480-487 (2005); Ying, Q. L., et al., Nat. Biotechnol., 21(2):183-186 (2003)), most commonly in conjunction with embryoid body formation, improved control and scalability during the differentiation process can further enhance current methodologies. Therefore, in order to control differentiated cell output, a more complete understanding of the factors that regulate lineage commitment needs to be defined.
Mesenchymal stromal cells (MSCs) have shown therapeutic benefits in models of GVHD, myocardial infarction, fulminant hepatic failure, central nervous system trauma and others. MSCs reduce tissue inflammation in many traumatic or inflammatory disorders and thereby secondarily effect tissue repair. Researchers have suggested that MSCs, through soluble factor secretion instead of direct cell replacement, orchestrate cascades of biochemical cues which both mitigate fibrosis and promote tissue protection. These advances have propelled tissue protective MSCs to the forefront of cellular therapeutic development. An increasing interest has also been drawn to the capability of transplanted MSCs to improve SCI outcomes via secretion of cytokines and neurotrophic factors (Eaves, C. J., et al., Blood, 78, 110-117 (1991), Himes, B. T., et al., Neurorehabil. Neural Repair, 20, 278-296 (2006), Parekkadan, B., et al., PLoS ONE, 2, e941 (2007) et al.), which may both reduce inflammation and promote neural cell growth and differentiation. Spinal cord injury (SCI) involves a primary mechanical injury followed by a series of cellular and molecular secondary events resulting in progressive destruction of spinal cord tissue. Functional deficits following SCI result from damaged axons, loss of neurons and glia, and demyelination, whereas the inflammatory reaction contributes to marked apoptosis and scar tissue formation, thereby preventing axon extension and re-establishment of appropriate neuronal connections.
Alginate, a biocompatible copolymer of mannuronic and guluronic acid, has been used for many cell and tissue engineering applications, including, to mature hepatocyte function, to encapsulate embryoid bodies, to promote EB differentiation and to induce MSC differentiation (Magyar, J. P., et al., Ann. NY Acad. Sci., 944, 135-143 (2001), Steinert, A., et al., J. Orthop. Res., 21, 1090-1097 (2003), Sun, A. M., et al., Appl. Biochem. Biotechnol., 10, 87-99 (1984)), or to direct embryonic stem cells towards hepatocyte lineage (Maguire, T., et al., Biotechnol. Bioeng., 98, 631-644 (2007), Maguire, T., et al., Biotechnol. Bioeng., 93, 581-591 (2006)). Although studies have indicated that the microenvironment as well as the developmental status of MSCs can alter neural stem cell inductive signals (Croft, A. P. and Przyborski, S. A., Exp. Neurol., 216, 329-341 (2009)), MSC tissue persistence, potential MSC differentiation and/or MSC migration away from the injury site are very complex and present dynamic problems, which are difficult to resolve, control and quantify.
In particular, several drawbacks in current MSC implantation approaches limit safe and controlled clinical trial implementation. These include, 1) directly transplanted MSCs exposed to the complex injury environment may be adversely affected early in the treatment process, 2) MSCs may migrate to undesired tissue locations, and 3) MSCs may differentiate into undesired end stage cells. These issues severely limit the development of controlled feasibility studies and ultimately translatability of MSC treatments into clinical settings. Many experimental variables of MSC use have not been thoroughly evaluated including molecular mechanism(s) of anti-inflammatory MSC function. Furthermore, recent findings have identified donor specific phenotypic MSC differences, further necessitating controlled approaches for cell delivery. Therefore, engineered methods for controlled MSC delivery, without comprising their tissue protective properties, must be developed, and there remains a need in cell replacement therapies using renewable stem cell sources such as MSCs to treat a wide range of degenerative diseases.
Moreover, although studies have established techniques to successfully differentiate stem cells into different mature cell lineages using growth factors or extracellular matrix protein supplementation in both two and three-dimensional configurations, their practicality is limited by lack of control and low yields of differentiated cells. In particular, engineered methods for controlled MSC delivery, without compromising their tissue protective properties, must be developed, and there remains a need in cell replacement therapies using renewable stem cell sources, such as embryonic stem cells and MSCs, to treat degenerative diseases or disorders.